The cell-specific expression of gonadotropin-releasing hormone (GnRH) is essential for the coordinate regulation of the mammalian reproductive system. Regulation of GnRH neuronal activity has been difficult to study due to their scattered distribution and paucity of cells. Immortalized neuronal cell lines created by targeted tumorigenesis in the mouse (NLT and Gn11) have been shown to secrete variable amounts of GnRH, with NLT cells producing about ten times higher levels of GnRH than Gn11 cells. Comparison of these cell lines, therefore, provides a means of identifying the factor or factors that may be responsible for cell-specific expression of GnRH. A subtraction hybridization technique using NLT and Gn11 mRNA has been used to isolate a cDNA clone that is differentially expressed in NLT cells, designated NLT expression factor 1 (NLT-EF1). Northern blot analysis is differentially expressed in NLT cells, designated NLT expression factor 1 (nNLT-EF1). Northern blot analysis confirms differential expression, revealing three-fold higher levels of NLT-EF1 mRNA present in NLT cells when compared to Gn11 cells, and identified NLT-EF1 in hypothalamus, cortex, cerebellum, lung, and testis. When compared to Gn11 cells, and identifies NLT-EF1 in hypothalamus, cortex, cerebellum, lung, and testing. When expressed in Gn11 cells, NLT-EF1 increase GnRH gene expression, suggesting a regulatory effect of GnRH gene expression. Utilizing the subtraction hybridization, as well as an mRNA differential display technique, the identification of other novel cDNAs is proposed, as well as investigation of their role in GnRH gene regulation. This includes anatomic co-localization with GnRH as well as structural and functional studies of putative factors. In addition since the cell specific element of the GnRH gene has been isolated, a yeast one-hybrid technique will also be used to screen a mouse hypothalamic cDNA library to isolate GnRH neuronal specific transcription factors. These will also be further characterized as above. The precise coordinated expression and release of GnRH is essential for a functioning mammalian reproductive system, yet the factors that control GnRH gene expression remain largely unknown. The proposed techniques will allow the identification of factors responsible for cell-specific GnRH expression and regulation. The utilization of multiple techniques for the identification and characterization of novel proteins important in gene regulation will allow me to develop the skills necessary to become an independent investigator.